This project is concerned with analyses of the genetic diversity of medically important parasitic protozoa and its implications to the epidemiology, course, and diagnosis of disease. The project has become increasingly involved in elucidation of diversity at the molecular level. Due to circumstances beyond our control, we have terminated several ongoing projects described in previous reports and have concentrated our efforts in three areas: (1) the detection and elucidation of intra- specific differences in Trypanosoma cruzi. Using the RAPD assay we have identified an oligonucleotide which differentiates between stocks of T. cruzi at the DNA level. Two unique DNAs of 279 and 290 bp have been isolated and are partially characterized. (2) We are attempting to apply the mathematical modeling methods we developed for analyses of the DNA synthetic cycle of Entamoeba spp. to Giardia lamblia. The method involves the use of a computer model to decompose flow cytometer-derived DNA histogram into their G1, S, and G2 components. (3) We have developed low-light-level video microscopy methods usable for studies of the role of the spleen in a malaria infection. Even though the spleen is known to be a site of parasite clearance, the mode of action is not known. We use a fluorescent lipophilic dye to mark the membrane of parasitized erythrocytes and a fluorescent monoclonal antibody to mark the marginal macrophages of the spleen. Images of each reaction are captured into a computer and merged electronically to evaluate the role of the spleen in a malaria infection.